Ultrasensitive Direct Fluorescent Immunoassay for Thyroid Stimulating Hormone

Phycobilisomes are photosynthetic antennae complexes of red algae and cyanobacteria (1)(2)(3) . They have been chemically cross-linked in such a way that they remain soluble and stable (4) . These stabilized phycobilisomes (PBXLTM dyes) have large complex weights (between 1.0 × 107 and 1.5 × 107 Da) and Stokes shifts. They contain a large number of chromophores coordinated to efficiently transfer energy down an energy gradient and emit between 662 and 666 nm. The PBXL-1 dye, used in the thyroid-stimulating hormone (TSH) model, contains B-phycoerythrin, R-phycocyanin, and allophycocyanin as its component phycobiliproteins. Each PBXL supramolecular complex can deliver up to 1400 chromophores per binding event without indirect signal generation steps, signal amplification, or enzyme substrates. PBXL dyes provide physical amplification of signal, enabling ultrasensitive direct fluorescent immunodetection of such clinically relevant analytes as TSH. TSH was used as a model system for a microplate immunoassay because of its clinical importance and high sensitivity requirements. The detection limit of the PBXL based assay was 0.01 mIU/L (6.2 × 10−14 mol/L). We used the following reagents: plate-coating buffer containing 100 mmol/L sodium phosphate (pH 7.4), 150 mmol/L sodium chloride, and …